Peptide‐functionalized double network hydrogel with compressible shape memory effect for intervertebral disc regeneration

Abstract As a prominent approach to treat intervertebral disc (IVD) degeneration, disc transplantation still falls short to fully reconstruct and restore the function of native IVD. Here, we introduce an IVD scaffold consists of a cellulose‐alginate double network hydrogel‐based annulus fibrosus (AF) and a cellulose hydrogel‐based nucleus pulposus (NP). This scaffold mimics native IVD structure and controls the delivery of Growth Differentiation Factor‐5 (GDF‐5), which induces differentiation of endogenous mesenchymal stem cells (MSCs). In addition, this IVD scaffold has modifications on MSC homing peptide and RGD peptide which facilitate the recruitment of MSCs to injured area and enhances their cell adhesion property. The benefits of this double network hydrogel are high compressibility, shape memory effect, and mechanical strength comparable to native IVD. In vivo animal study demonstrates successful reconstruction of injured IVD including both AF and NP. These findings suggest that this double network hydrogel can serve as a promising approach to IVD regeneration with other potential biomedical applications.


| INTRODUCTION
Degenerative disc disease (DDD) entails the breakdown of one or more intervertebral discs (IVD). Common symptoms include disc bulging, herniation, and the formation of osteophyte (bone spurs). These conditions often interfere with nervous structures and cause pain in back or neck which can eventually lead to peripheral neuropathy. 1,2 Causes for disc degeneration are numerous, such as aging, acute overloading, or long-term stress. These factors often result in progressive decline in disc nutrient supply and changes in extracellular matrix (ECM) composition, thereby weakening tissue strength and altering cellular metabolism. 3 Unfortunately, the self-repair ability of IVDs is poor, and the degeneration process is often irreversible. 4 Surgical interventions to remove the injured/degenerated IVD and replace with artificial device are often applied when the disc can no more maintain cellular activity and biochemical environment.
Although total disc replacement (TDR) has been regarded as a promising approach to reduce pain and reverse degeneration, the implanted scaffolds still have several drawbacks. First, compared with human cortical and cancellous bone, metallic TDR possesses extremely high elastic modulus, which can lead to the occurrence of stress-shielding effect between implantation site and intervertebral body. 5,6 Second, polymer-based scaffolds often fail to maintain integrity under constant compressive loading. 7 Failure to recover to full disc height runs the risk of material leakage. Third, low wear resistance of polymer increases the risk of osteolysis over long-term implantation. 8,9 Moreover, the invasive surgical procedure during implantation itself also raises a challenge.
Insertion of artificial discs often requires surgical approaches both anteriorly and posteriorly to the intervertebral space, posing high risk of complications and resulting in prolonged recovery time in patients. 10 Facing these issues, the aim of this research is to overcome the mechanical and biochemical limitations of conventional IVD implants and simplify the surgery to minimize the risk of morbidity.
Double network (DN) hydrogel has been introduced to strengthen and toughen the polymeric scaffold. It consists of two different types of network structure: one chemically crosslinked network and one physically crosslinked network. 11 In the former structure, polymer chains form a rigid bulk for DN hydrogels and maintain the shape. The later structure fills in the rigid network and absorbs excess energy. 12 Admittedly, physically crosslinked network has a less stable structure, unlike its chemically crosslinked counterpart. However, hydrogel formed by non-covalent bonds can usually undergo reversible process, which the bonding can immediately recover after breaking. 13 One major challenge that current polymer-based hydrogel faces is to fully replicate the biomechanical properties required to support multiaxial spinal loads. The elastic modulus of native IVD is about 0.5 to 1 MPa. Through the design of DN hydrogel, the polymer scaffold can enhance its mechanical strength to meet the needs of engineered discs.
Shape memory polymers (SMPs) are smart materials capable of recovering to a pre-determined shape in response to external stimuli. 14,15 Such property could be applied to simplify the implantation procedure as minimally invasive surgery. Besides, SMP scaffolds exhibit a self-fitting capacity from the force generated during shape recovery process. 16 The capacity ensures the implanted hydrogel to tightly fit in the original space of degenerative disc. The rigid contact can prevent material sliding by forming a constraint force between the scaffold and adjacent vertebrae.
Mesenchymal stem cells (MSCs) play an important role in tissue regeneration from their abilities to sustain long-term self-renewal and differentiate into other cell types. 17 Critically, MSCs engage by migration to the wound when injured. 18 By mimicking the configuration of bone marrow homing peptide (BMHP), a short peptide sequence SKPPGTSS (SKP) has been reported to facilitate MSC homing. 19,20 With the engraftment of SKP peptide, the hydrogel serves as a platform for MSCs homing. Meanwhile, for supporting in-growth and adhesion of endogenous stem cells, cell adhesion peptide sequence GRGDSP (RGD) is designed and immobilized on polymer network to augment cell/matrix interactions. 21,22 Various growth factors have been proposed in vitro to stimulate ECM synthesis by IVD cells, including transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), and bone morphogenetic protein-2 (BMP-2). 23,24 However, the angiogenic potential of the growth factors (TGF-β or IGF-1) may adversely promote blood vessel ingrowth, which is undesirable in degenerative disc treatment. 25 On the other hand, Growth Differentiation Factor-5 (GDF-5) receptors are non-angiogenic, and GDF5 gene was shown to be a susceptiblity gene for disc degeneration, suggesting that GDF-5 can be a promising candidate to stimulate ECM. 26 Also, GDF-5 was reported to enhance the proliferation of both AF and NP cells, and to down-regulate the metalloproteinase (MMP) expression by inhibiting ECM catabolism. 27 However, current trends of delivery of GDF-5 via single injection may breakdown the disc and thus compromise the long-term outcome.
Here, we aimed to develop a tough and bioactive hydrogel to replace the degenerated discs. A highly compressible DN hydrogel was synthesized via physically crosslinked alginate network and chemically crosslinked cellulose network (Figure 1a). While DN hydrogel was made into cylinder shape, serving as the AF, the NP was formed via the injection of prime cellulose solution into the central part of the scaffold. With structural mimicry of native IVD, this scaffold can provide therapeutic effect on both NP and AF. Shape memory property was also incorporated by addition of chelating agents, and the DN hydrogel could underwent compression and recovered the disc height, indicating the feasibility of minimally invasive surgery (Figure 1b). Furthermore, MSC homing peptide (SKP peptide) and cell adhesion peptide (RGD peptide) were modified on the polymer chain of cellulose and alginate, respectively ( Figure 1c). The SKP peptide can recruit endogenous MSCs toward the injured site and the RGD peptide can enhance cell survival and attachment. Meantime, the MSCs can benefit from GDF-5, which is released from the central part of scaffold, and differentiate into NP-like cells, contributing to ECM formation and proteoglycans synthesis. Collectively, we believe that the reported hydrogel will serve as a promising alternative for total disc replacement and delay discs degeneration in combination with other treatment modalities.

| Functional peptide sequences modification
Peptide-functionalized cellulose was prepared in accordance with the method described in previous study. 36 In brief, 0.4 g of cellulose microcrystalline powder (Alfa Aesar, USA) and sodium periodate were added to 20 ml of deionized water, protected from light. After 24 h, the reaction was stopped by adding 5 ml ethylene glycol to the solution and stirring for 1 h. The reaction mixture was purified by centrifugation with deionized water to remove excess ethylene glycol.
To prepare peptide-functionalized cellulose, Schiff base reaction was conducted between aldehyde groups of cellulose-ALD and amino groups on the glycine of peptide sequence. In brief, 3 mg of GGSKPPGTSS (SKP peptide; Allbio, Taiwan) was dissolved in 5 ml deionized water. The solution was carefully titrated, and the pH value was adjusted to 8 by 1 N NaOH. 50 mg of cellulose-ALD was added to the solution, and the mixture was stirred at 40 C for 4 h. The resulting solution was then centrifuged with deionized water for three times to remove the unreacted SKP peptide followed by lyophilization for further storage.
As for the preparation of peptide-functionalized alginate, the method was described in previous study. 37 Shortly, 50 mg of sodium alginate (Alfa Aesar) was dissolved in a 2-(N-morpholino) ethanesulfonic acid (MES) buffer at room temperature (pH = 6.5, 0.3 M NaCl). The final solution was lyophilized and stored at À20 C for further use. The modification of functional peptide sequences was determined by 1 H NMR (Bruker, AVANCE-500) and FT-IR (Bruker, Vertex 80v).

| Preparation and characterization of DN hydrogel and total IVD scaffold
Alkali/urea solvent system was utilized to dissolve cellulose-SKP powder. 38 In brief, cellulose-SKP was added to 10 ml of alkali/urea solution (0.8 g NaOH and 0.4 g Urea in 10 ml deionized water) with vigorous stirring. After 24 h storing under À80 C, the frozen solution was thawed at room temperature. When the solution was completely thawed, it was placed the mixture back to À80 C again. The freezethaw cycle was repeated for three times, and a completely dissolved cellulose solution was obtained. Next, alginate-RGD powder was added into previous cellulose solution. Epichlorohydrin Figure S8). For the total IVD scaffold preparation, the center part of cellulose/alginate DN hydrogel was removed by a biopsy punch, forming a hollow cylinder. Then, cellulose solution was injected into the inner part until gelation; thus, a total IVD scaffold with inner and outer parts was synthesized.
Scanning electron micrographs were taken with a high-resolution thermal field emission scanning electron microscope (SEM; JEOL, JSM-7610F). Both cellulose hydrogel and DN hydrogel were frozen in liquid nitrogen and snapped immediately. The frozen samples were then immersed in adequate amount of water and stored in À80 C for 24 h. After completely freezing, the samples were lyophilized consequently. The fracture surface of the hydrogel was sputtered with gold, observed, and photographed.
Swelling ratio of the hydrogels was measured at room temperature. The hydrogel samples were incubated in 1Â phosphate-buffered saline. Both the volume and weight of each hydrogel sample were measured before and after the incubation with PBS at predetermined time points. The swelling ratio of hydrogel is defined as:

| Mechanical property test
Mechanical behaviors were tested by compression test using a universal testing machine (UTM, Instron 3400). A cylindrical hydrogel (about Ø 14 mm Â 9.5 mm) was prepared as described above. Then, the hydrogel was loaded on the lower plate and compressed by the upper plate at a strain rate of 1 mm min À1 . All samples were analyzed to obtain the stress-strain curve. As for cyclic test, total IVD scaffold was tested by repeated loading to 50% strain. Then, 20 cycles of loading and unloading were done to the hydrogel with a strain rate of 3 mm min À1 . All samples were measured at room temperature, and the data was further analyzed using Bluehill 3 software.

| MSC proliferation and differentiation assessment
The IVD scaffolds with or without GDF-5 protein incorporation were fabricated and co-cultured with rMSCs. To determine the cell proliferation, the amount of WST-8 was quantified using Cell Counting Kit-8 (CCK-8 Assay; Abcam). 50 μl of hydrogel samples were placed in 48-well plate initially. 0.5 ml of culture medium (2.5 Â 10 4 cells/well) was carefully added to each well and changed by 50% every 2 days.
After incubating for 1, 3, 7, and 14 days, 50 μl of CCK-8 solution (1/10 of medium volume) was added to each well. The microplate was then set in the incubator for 2 h until the color changed to orange.
Absorbance at 450 nm was measured using SpectraMax Plus384 microplate reader.
The impact of GDF-5 protein on rMSCs was determined by the expression levels of marker genes of chondrocytes and NP-like cells.

| Cell migration test
In order to verify the recruitment of rMSCs, inserts with a diameter of For fluorescent staining, rMSCs were fixed with 4% formaldehyde at 4 C for 30 min and treated with 0.1% Triton X-100 for 5 min. The cellular nucleus was further stained with 4 0 ,6-diamidino-2-phenylindole (DAPI, blue) (10 μg ml À1 ) for 10 min in dark. As for H&E staining, the inserts were placed into 4% formaldehyde for 5 min at room temperature to fix the cells on the membrane. The membrane was then stained with hematoxylin solution for 5 min and washed in tap water followed by stained with 1% eosin (Gills, USA) for 1 min, following washing in tap water. After the staining process, the inside of each insert was gently swabbed using cotton swabs, taking care not to damage the membrane or touch the underside of the inserts. The Transwell inserts were placed under fluorescent microscope and optical microscope to count cell numbers of the selected visual fields (5 visual fields/insert, 10Â).
Direct observation of Transwell insert membrane was also conducted with the help of SEM. Medium in the inserts was removed and gently rinsed with PBS, following with fixation in 4% formaldehyde for 30 min at 4 C. The membrane was carefully removed from the insert using a scalpel blade and mounted onto the silicon wafer for further preparation. The samples were then sequentially dehydrated with 50% ethanol for 10 min, 75% ethanol for 10 min, 95% ethanol for 10 min, and immersed in 100% ethanol for 10 min (twice). Afterwards, critical point drying (CPD) was performed, and the membrane was further sputtered with gold.

| Cell adhesion test
Hydrogel samples were prepared, placed in a 48-well plate, and incubated at 37 C, 5% CO 2 for 10 min. 20 μl of rMSC suspension solution

| Animal and surgical procedures
The experimental protocols were approved by the Institutional Animal  Table S2. The following surgical procedure for degenerated disc disease was performed. 39,40 After the preparation of total IVD scaffolds containing functional peptide sequences and bioactive molecules, the artificial discs were implanted into rats as caudal spine discs between third and fourth caudal vertebrae (caudal 3/4) to evaluate their long-term performance. Rats were anesthetized using zoletil (Zoletil 50, 50 mg ml À1 ; Virbac, France) (30 mg kg À1 ), and xylazine 7.5 (mg kg À1 ) (Rompun, 20 mg ml À1 ; Bayer Korea, Korea), which were mixed together and administered intramuscularly. An initial dose of 25 mg kg À1 cefazoline (Standard Chem & Pharm, Taiwan) was injected intramuscularly after anesthetic injection. Native disc was removed by a rongeur, and a disc space was prepared for implant insertion in the tail. The vertebral column was later exposed allowing insertion of the IVD scaffold into the original space. The disc space was released to press-fit the implant in place and wound closure was performed with 3-0 surgical suture. There was no animal died during the experiment period.

| Magnetic resonance imaging (MRI) analysis
For MR scanning, rats were anesthetized with isoflurane (1.5-2%) and placed in 3.0 T MRI scanner (Philps, Ingenia 3.0 T). The rat tail was held by tape to prevent movements ( Figure S9). T2-weighted images and disc degenerative degree of each group were quantified by the high signal ratio in IVD using the Image J software. 41 The high signal ratio is defined as: High signal ratio in disc ¼ High signal area of experimental condition High signal area of adjacent normal disc Pfirrmann et al. introduced a grading system for disc degeneration based on MR signal intensity including disc structure, distinction between nucleus and anulus, and disc height. 42,43 In this study, we modified this grading system in order to properly evaluate the regenerative effect of our scaffold. The grading scale was shown in Table S3.

| Histological and immunohistochemical examination
Rats were deeply anesthetized using 5% isoflurane and sacrificed.
Discs as well as adjacent vertebrae were extracted and then fixed in 4% phosphate-buffered paraformaldehyde at 4 C for 2 days followed by washed in running tap water overnight to remove residual formalin.
The samples were then sequentially decalcified in DECALCIFIER II  Table S4. 44,45 AbPr staining was conducted by treating the de-paraffinized cryosections with Alcian blue solution for 15 min followed by rinsing in running tap water for 10 min. Then, the cryosections were incubated with Picrosirus red solution. After staining for 45 min at room temperature, the samples were rinsed in 0.5% aqueous acetic acid for 5 min.
The final product was then sequentially dehydrated in 95% ethanol and 100% ethanol (twice) for 2 min and mounted on the glass slide.
H&E staining samples were prepared as previously described. The stained samples were observed and recorded with optical microscope.

| Biomechanical property test
Motion segments and intact native motion segments were both cleaned with surrounding tissue to result in bone-disc-bone motion segments after sacrificing the animals at 8 weeks. The mechanical property of the IVD implants at the moving segment, consisting of the IVD and the adjacent vertebrae, was tested using UTM at a compression rate of 0.5 mm min À1 . The height of the sample was 12 mm and the diameter of the IVD was 10 mm.

| Statistical analysis
Data were presented with mean ± SD. Statistical significance was Though the total IVD scaffold showed hysteresis loops after cycles of loading and unloading, there were no considerable changes within 40 cycles. These results indicated that the total IVD scaffold could maintain its integrity and recover to its original shape without permanent deformation after subjecting to cycles of continuous compressive loading and unloading (Movie S1).

| Shape memory properties of DN hydrogel
The design strategy for shape memory hydrogel primarily involves two distinct components: permanent crosslink which determines the permanent shape, and reversible crosslink which fixes its temporary shapes. In our DN hydrogel, the covalent bonds in cellulose network may act as permanent crosslink, while the noncovalent bonds in alginate network serve as reversible crosslink.
Owing to the combination of cellulose and alginate network, shape memory property could be observed after the addition of ethylenediaminetetraacetic acid (EDTA) chelating agent. EDTA has been widely used in To quantify the shape memory behavior of the DN hydrogel more precisely, the parameters of shape fixity and shape recovery ratio were measured ( Figure 4b). As the compressive strain of double network hydrogel was set to 10% and 20%, the shape fixity ratio and recovery ratio were over 95%. The two parameters were still higher than 85% even when the compressive strain was increased to 30%.
The explanation for slight decrease in the recovery ratio was the buckling of hydrogel network during deformation. Since there was low physical crosslinking density between alginate network with calcium ions, the compressed hydrogels were unable to fully recover to their original shape. Such phenomenon could be adjusted by modulating physical crosslinking density for future clinical requirements.

| In vitro controlled signaling regulation of MSCs
To confirm the possibility of using the total IVD scaffold as structural support for injured disc replacement, the cytocompatibility of Since MSC proliferation is an important factor in endogenous IVD regeneration, we first evaluated the ability of growth factor GDF-5 by CCK-8 assay (Figure 5a). MSCs were co-cultured with total IVD scaffold either with or without GDF-5. In GDF-5 loaded scaffold at day 7, the relative proliferation rate tended to be higher when compared with pristine scaffold group, although this difference was not signifi-

| In vitro MSC recruitment and adhesion
In order to examine the locally directed homing capability of SKP-

| In vivo degenerative disc disease treatment in rat model
The therapeutic efficacy of total IVD scaffold was evaluated using a rat caudal disc model. The scaffolds were implanted into the caudal spine discs of the rats, between the third and fourth caudal vertebrae (caudal 3/4), to evaluate their long-term performance (Figure 7a). The shape of the scaffolds was fashioned to fit the space of rat caudal disc ( Figure 7b). After removing the attached peripheral connective tissue, F I G U R E 5 Effect of GDF-5 on MSC proliferation and differentiation. (a) The relative proliferation rate of MSCs cultured in total IVD scaffolds with and without loaded GDF-5 protein at different time points. Error bars show mean ± SD for total n = 4. (b-e) Gene expression levels of MSCs cultured in total IVD scaffolds with and without GDF-5 protein at different time points. GAPDH served as housekeeping gene. All genes were normalized to GAPDH levels. Error bars show mean ± SD for total n = 4 (*p < 0.05 and **p < 0.01) native caudal disc was found between two adjacent vertebrae ( Figure 7c). Native caudal discs were subsequently removed and the endplate of the vertebrae was carefully preserved. The space was then replaced with injection of prepared scaffolds or saline. Four and eight weeks after scaffold implantation, MRI images were obtained to determine disc height and water content in the IVD scaffolds ( Figure 7d). The abbreviation of each experimental group was described in Table S2. The GPDN group showed a fusiform shape with bright signal between the adjacent vertebrae. Similar result was also found in PDN group, indicating that both groups maintained their integrity and water content. Although DN group gave bright signal, the height of scaffold was relatively lower than that of negative control group (NC). On the other hand, no signal was found in DC group, resulting from the absence of any scaffold. To evaluate the disc height and integrity in detail, the area of high signal ratio between implanted scaffold and adjacent native disc was calculated and shown in cytotoxic concerns when added to biological samples. 28,29 Of note is that the exposure time of EDTA can be controlled in our study to minimize the cytotoxic effect. From the Movie S2, it should be noted that the hydrogel was recovered to the permanent shape within 1 min, indicating that the EDTA injecting time could be decreased to <5 min.
Furthermore, a lower concentration of EDTA solution could be considered when inducing the shape recovery process, thus the concern for the EDTA cytotoxic issue was minimized.
One of the important strategies for regulating MSCs is the deliv- (3) maintaining the scaffold integrity to withstand the constant mechanical loading in disc space. The implanted total IVD scaffold was demonstrated to meet all three requirements in rat caudal disc with the help of functional peptide modification and the encapsulation of growth factor GDF-5. This study provided unique evidence that the total IVD scaffold has great potential to replace the injured IVD in the spine. It is interesting to note that a previous research has reported distinct IVD degenerative changes and fibrotic healing in response to IVD injury between sexes. 33 Correlational network analyses showed that males demonstrated clear relationships between injury, structural IVD degeneration, and mechanical allodynia, while females did not. Thus, subtle sex differences in the spine likely interact with nervous system differences. Another research study also pointed out that pain transduction and the development of chronic pain differ between males and females. 34 Female exhibited increased sensitivity to nerve root injury, suggesting sex differences may be present in an IVD degeneration-related pain model. Insights from the above works suggest that it is necessary to treat female and male F I G U R E 8 Chondrogenic matrix production, organization, and histological score. Error bars show mean ± SD for total n = 3 (*p < 0.05, ***p < 0.001, and ****p < 0.0001 vs. negative control in Vimentin group; ## p < 0.01, and #### p < 0.0001 vs. negative control in FOXF1 group). (c) Plots of uniaxial compressive stress versus strain for different groups after 8 weeks of implantation animals as distinct cohorts. Further, there is also need for future work on low back pain models which examines how these spine and nervous system relationships may shift over time and across pain modalities.
A hydrated scaffold was observed within the disc space, maintaining the overall shape and disc height after 8 weeks of implantation under MRI inspection (Figure 7d). The integration with neighboring tissue could be observed in H&E staining results (Figure 8a). With the help of RGD peptide, continuous boundaries between scaffolds and vertebral bodies were found in both PDN and GPDN groups, while discontinuous boundaries were observed in DN group. Moreover, the production of proteoglycans and collagen in GPDN group was similar to healthy caudal disc, demonstrating that MSCs were successfully recruited to disc space and produced functional tissue de novo For future perspective, despite the successful implantation of the reported total IVD scaffold in caudal disc space, it is still different from actual clinical applications due to the differences between rat caudal disc and human lumbar disc. Since human beings are bipedal animals with upright standing, the IVD bears more axial loading and would require higher mechanical properties. Meanwhile, considering the larger size and relatively lower permeability of human IVD, the poor nutritional supply and fibrotic bioenvironment in disc space will potentially be a detrimental issue in human degenerative disc disease.
A tissue engineered cell/scaffold composite should possess a comparable mechanical strength with nature disc tissue to increase the longevity of implant. To address limitations, translating the IVD scaffold to rat lumbar disc or larger animal models will be our next follow-up study. In spite of these constraints, this study still introduces the formation of functional tissues in disc space by recruiting endogenous MSCs and inducing the differentiation of MSCs in vivo. The findings herein provide strong support for the development of functional IVD implants and evidence that the reported total IVD scaffold can serve as a promising candidate for future applications in IVD regeneration as well as for other biomedical applications in tissue engineering.

| CONCLUSIONS
In this study, we have successfully developed an IVD-mimicked and peptide-functionalized scaffold using cellulose/alginate DN hydrogel for annulus fibrosus (AF) layer and cellulose hydrogel for nucleus pulposus (NP) layer, respectively. The usage of DN structure promotes the mechanical properties of implanted IVD scaffold, which is essential for preventing further damage by adjacent tissue due to mechanical mismatch. Moreover, through the presence of periphery structure, the IVD scaffold allows the local preservation and sustainable release of GDF-5 growth factor in response to hydrogel degradation. The trigger of shape recovery process by addition of chelating agent enables the scaffold implantation to be done without intense and complicated surgical procedure. In cytocompatibility assessment, the composition of IVD scaffold did not show any toxicity to MSCs when incubated with hydrogel matrix. GDF-5 released from the IVD scaffold retained its bioactivity for MSC differentiation, as determined by qPCR. The chemotactic migration of MSCs toward the injured site was significantly augmented by the release of SKP peptide, while the RGD peptide-grafted hydrogel provided platform for the enhancement of cell survival as well as proliferation. In the study of rat caudal disc degeneration model, implantation of structure support through IVD scaffold together with the local delivery of GDF-5 and functional peptide sequences synergistically retarded the degeneration rate and subsequently enhanced functional recovery. These results demonstrate a potential to control the delivery of bioactive factors while facilitating the chemotaxis and proliferation of endogenous MSCs for IVD regeneration. The findings herein provide support for the development of functional IVD implants and evidence that the challenges associated with artificial disc may be overcome in the near future.

ACKNOWLEDGMENTS
We thank for the technical assistance from IBEN Service in National Health Research Institutes for MR scanning. We are also grateful to the help of Instrument Center in National Tsing Hua University

CONFLICT OF INTEREST
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

DATA AVAILABILITY STATEMENT
The raw data required to reproduce these findings are available to download as requested. The processed data required to reproduce these findings are available to download as requested.